FLP recombinase encoded by yeast (Saccharomyces cerevisiae) 2 μ DNA is a site-specific DNA recombinase that recognizes a particular DNA sequence of 34 nucleotides, which is referred to as FRT sequence, and performs an entire process of cleavage, exchanging and binding of a DNA strand between two FRT sequences, and binding [Babineau et al., J. Biol. Chem., 260, 12313-12319 (1985)]. When two FRT sequences having an identical orientation exist within the same DNA molecule, a DNA sequence flanked by the two FRT sequences is excised by the FLP recombinase, to form a circular molecule (excision reaction). On the other hand, when two FRT sequences exist in the different DNA molecules, one of which is a circular DNA, the circular DNA is inserted into the other DNA molecule via the FRT sequences (insertion reaction).
The insertion reaction and the excision reaction are reversible. However, when two FRT sequences exist within the same DNA molecule, by the insertion reaction, the excision reaction also takes place immediately thereafter. Therefore, the reaction equilibrium leans towards the side of the excision reaction. Therefore, it has been known that the frequency that a given DNA can be inserted into the other DNA molecule by the insertion reaction is very low.
FRT sequence consists of a DNA sequence of 34 bp [Jayaram et al., Proc. Natl. Acad. Sci. 82, 5875-5879 (1985)], wherein a sequence of 8 bp flanked by two inverted repeats of 13 bp is referred to as a spacer region. It has been known that DNA recombination is carried out in the spacer region [Umlauf S. W. et al., EMBO Journal, 7, 1845-1852 (1988); Lee J. et al., EMBO Journal, 18, 784-791, 1999]. FRT sequence (SEQ ID NO: 1) is shown:
123456785′-GAAGTTCCTATACTTTCTAGAGAATAGGAACTTC-3′spacer region
It has been found that by changing the nucleotides of the spacer region with nucleotides which are different from inherent FRT sequence (wild-type FRT sequence), i.e. mutant FRT sequence, specific DNA recombination takes place between two mutant FRT sequences but no specific DNA recombination reaction takes place with the wild-type FRT sequence [Schlake T. et al., Biochemistry, 33. 12746-12751(1994)]. Further, it has also been shown that genes existing on two different DNA molecules can be replaced in the presence of FLP recombinase by using this mutant FRT sequence in cultured animal cells. In other words, it has been shown that a gene A existing between the mutant FRT sequence and the wild-type FRT sequence on a given DNA molecule can be replaced with a gene B existing between the mutant FRT sequence and the wild-type FRT sequence on the other DNA molecule [Schlake T. et al., Biochemistry, 33, 12746-12751 (1994); Seibler J. et al., Biochemistry, 36, 1740-1747 (1997)].
A sequence (referred to as “F3,” SEQ ID NO: 6) having TATTTGAA in the spacer region has been known as one of known mutant FRT sequences resulting from introduction of a mutation in the spacer region of FRT sequence (Seibler J. et al., supra). Seibler et al. conducted gene replacement on a chromosome of an animal cell using this mutant FRT sequence (F3) and a wild-type FRT sequence. However, the efficiency of gene replacement was as low as 21 to 38% even though cells in which a gene was replaced were enriched by drug selection using a drug-resistance gene before and after gene replacement [Seibler J. et al., Biochemistry, 37, 6229-6234 (1998)]. It is thought that this gene replacement efficiency is further lowered if the drug selection is not carried out. In other words, the mutant, F3 of the prior art is an insufficient sequence for performing highly efficient gene replacement reaction, so that a more efficient mutant FRT sequence is desired.
Also, the gene replacement efficiency using the mutant FRT sequence (referred to F5, SEQ ID NO: 7) having the spacer region, CTTGTGAA) is not sufficient in actual use.